"Blood vessels go where they are needed", see A. A. Liebow in Handbook of Physiology, Section 2: Circu ation, vol. 2, p. 1251-1276, Amer. Physiol. Soc. Washington, 1965. The homeostasis of the body and its organs and tissues depends on regulatory mechanisms of angiogenesis (lateral and directional growth of blood vessel capillaries). This homestasis of blood vessel patterns influences tissue repair and wound healing, tissue neoformation in embryonogenesis and in reproductive cycles as well as survival, out-growth, regression and destruction of tumors, grafts and non-vascularized tissues. All these are examples of tissue regeneration processes.
It has been assumed that, as morphogens, soluble mediators may induce and regulate neoformation of blood vessels upon accumulation of leukocytes in the course of inflammatory processes; see A. A. Leibow, loc. cit.; W. Schaper (ed.), Pathophysiology of Myocardial Perfusion, Elsevier-North Holland, Amsterdam 1979; H. I. Peterson, (ed.) "Tumor Blood Circulation" CRC Press, Boca Raton 1979, with further references.
The destruction of tissue in inflammations caused by non-immunological and immunological processes induces the formation of different endogenic substances (mediators and hormones). They regulate the complex steps of activation of the inflammation and tissue regeneration processes. The mediators are formed either by limited and regulated proteolysis of plasma and serum protein factors as humoral mediators; or they are liberated by active secretion and/or cell lysis from cells and tissues as cellular mediators. Especially the mediators and hormones are important as specific carriers of chemical information which are formed and secreted by leukocytes in the course of cell proliferation processes (mitosis processes). They are components of the body's defence system whose systemic and local activation they regulate. The mediators contribute to the removal and detoxification of destroyed body's own components and/or intruded foreign components. In addition, by regulation of cell proliferation and tissue growth processes in wound-healing, they contribute to the restoration of physiological functions of the organism. As the classical hormones of endocrine glands, inflammatory mediators are trace components of tissues or blood and are present in very minute concentrations only. Experimental evidence shows that only up to 5,000 of such mediator protein molecules can be maintained in a steady state equilibrium by a cell in the mitotic cycle in its surrounding medium.
The sprouting of blood vessels occurs by chemotropism. Chemotropism is a reaction by which the direction of hyperplastic or hypertropic growth of tissues or organisms is determined by chemical substances in the cellular environment. The growth can occur in direction to the substance along its increasing concentration gradient; or it can occur away from the substance along its decreasing concentration gradient. Accordingly, directional growth is called positive and negative chemotropism, respectively; see W. G. Rosen, Quart. Rev., Biol., vol. 37 (1962) p. 242 to 259 with further references.
Specifically acting, endogenous chemical self-components with mitogen activity (chemotropic mitogens) which cause chemotropism in blood vessels along their increasing concentration gradient, are called by definition "angiotropins". Angiogenesis is a common characterstic of most inflammation, tissue regeneration and tissue growth processes, such as those caused by bacterial infections, in tumors and heart muscle infarction. The first inducing steps are endogenous or exogenous tissue injury processes, such as ischemic and immunological tissue injury processes, respectively.
Numerous factors which themselves are not chemotropically active, may indirectly induce directional growth of blood vessels. They are called angiotropinogens. These factors include known tissue damaging substances, such as silver nitrate, sodium hydroxide and endotoxins; see C. H. Fromer et al., Amer. J. Pathol.. vol. 82, (1976), p. 157 to 170.
Such exogenous foreign substances as endotoxins have a strong indirect biological action on tissue systems; see O. Luderitz, Angew. Chemie 82 (1970), p. 708 to 722. It is known that, on the one hand, toxins activate blood plasma protein systems, such as the kinine and the complement protein systems. On the other hand, they display mitogenic activity on mononuclear leukocytes (B-cell-mitogen); see J. Andersson et al. J. Exp. Med. 137 (1973), p. 943 to 953.
The participation of soluble tissue and cell extracts in mechanisms leading to blood vessel growth has already been postulated; see Liebow, loc. cit. M. Klagsbrun et al., Cancer Res., vol. 136 (1976), p. 110 to 114, D. Tuan et al., Biochemistry, vol. 12 (1973), p. 3159 to 3165 and J. Folkman et al., J. Exp. Med. vol. 133 (1971), p. 975 to 988 have been able to prepare crude extracts from tumors. The composition of such complex mixtures of substances which are represented by crude extracts of tumor tissues has not been further characterized biologically or chemically. However, these authors have been able to show that such extracts can stimulate blood vessel growth. A molecular estimation of activity distributions of such complex mixtures of crude extracts of tissues suggested that the blood vessel growth stimulating activity may be associated with molecular weight distributions larger than 100,000 dalton. L. U. Mostafa et al., J. Path. vol. 132 (1980), p. 191 to 215 have shown that such crude tumor extracts are also chemotactically active for leukocytes. Thus, such extracts may also cause a leukocyte accumulation. This shows that the apparent effects induced by these crude tissue extracts are supposedly based on indirect mitogen action, namely by means of the accumulation of leukocytes. More recently, blood vessel growth-stimulating activity has been associated with low molecular weight products such as prostaglandins; see R. Baserga (ed.): Tissue Growth factors. Handbook of Experimental Pharmacology, Springer Verlag, Berlin 1981, with further references.
However, prostaglanidins form a class of complex unsaturated fatty acid derivatives which are known to cause non-specific inflammatory processes, and thus possibly indirectly angiogenesis; see G. Weissman, B. Samuelsson and R. Paoletti (eds.): Adv. Inflammation Res., Raven Press, New York 1980.
Accordingly, Polverini et al., Nature, vol. 269 (1977) p. 804 to 806 have shown that phagocytizing macrophages can produce blood vessel growth-stimulating activities of unknown nature. In addition, it has been found that granulocytes and lymphocytes may be also a source of such blood vessel growth-stimulating activities; see C. H. Fromer et al., loc. cit.; Peterson et al. loc. cit.
The possibility that blood vessel growth-stimulating activities stem from leukocytes accumulated at reaction sites of inflammation has also been suggested by several facts. Thus, for preparation of such crude soluble blood vessel growth-stimulating activities from tissues, various non-physiological conditions have been used. They include the extraction of tissues by organic solvents, the degradation of tissues by tryosin and exposure of tissue extracts to hydrochloric acid. It is known that such extreme conditions may cause non-physiological changes in the structure and activity of proteins. These changes in proteins may be associated with the expression of new biological activities which are not intrinsic characteristics of the proteins themselves. These biological effects include phlogistic activities in such structurally changed proteins; see J. H. Wissler in Proc. Immunosymposium Vienna 1973, "Gram-negative Bacterial Infections and Mode of Endotoxin Actions; Pathophysiological, Immunological and Clinical Aspects" (B. Urbaschek, R. Urbaschek and E. Neter (eds.), Springer-Verlag, Vienna 1975, p. 91-105.
The existence of cellular mediators which may cause directional growth of blood vessels, in a biologically specific manner and free of side effects, has not been known or shown so far.
Chemotropism of blood vessel sprout is measured by chemotropic neovascularization of the cornea of rabbits or guinea pigs after focal administration of the substance to be investigated. The rabbit cornea is a physiologically avascular, transparent tissue. Another assay to test blood vessel growth, vascularization of tissue and morphogenesis of blood vessel patterns is the chorioallantoic membrane assay by which the sprouting and formation of blood vessel patters in chicken embryos is investigated. A third assay investigates the mitogenic activity of the substance assayed on cultured endothelial cells.
It is therefore a primary object of this invention to provide a new class of cellular angiotropins from leukocytes.
It is another object of this invention to provide a new class of cellular angiotropins from leukocytes in highly purified, molecularly homogenous form.
It is another object of this invention to provide a new class of cellular angiotropins from leukocytes in physical quantities for practical use.
It is another object of this invention to provide a new class of angiotropins from leukocytes, which represent biologically specific, active and naturally acting mediators of angiogenesis reactions.
It is another object of this invention to provide a new class of chemorecruitins from leukocytes, which are capable of specifically inducing the directional growth of blood vessel sprouts in vivo.
It is still another object of this invention to provide a process for producing and obtaining a new class of angiotropins from leukocytes in a highly purified, molecularly homogenous form and in physical quantities for practical use.
It is still another object of this invention to provide a pharmaceutical composition for specifically inducing the directional growth of blood vessels.
These and other objects and advantages of the present invention will be evident from the following description of the invention.